HIV transgenic animals and uses therefor

ABSTRACT

The invention provides transgenic animals comprising a lentiviral transgene, such as an HIV transgene. Also within the scope of the invention are cells and eggs from the transgenic animal. Further included are methods for identifying therapeutic compounds for preventing lentiviral infection and treating associated disease (e.g. AIDS).

STATEMENT OF RIGHTS

This invention was made with government support under DE09170 awarded bythe National Health Institutes. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus (HIV) is an etiological agent of AcquiredImmune Deficiency Syndrome (AIDS). AIDS was first reported in the UnitedStates of America in 1981. As of January 1997, approximately 1.5 millioncases of AIDS in adults and children had been reported to the WorldHealth Organization (WHO); however, because reporting is difficult, WHOestimates that there were more than 8.4 million cases, about 580,000 ofwhich reside in the United States. However, the number of HIV infectedindividuals is much higher: as of January 1997, WHO estimated that therewere approximately 29.4 million HIV infected individuals world-wide,with about 1 million in the United States. It has been estimated that bythe year 2000, between 40 and 100 million individuals will be infectedwith HIV.

HIV, which has also been referred to as lymphadenopathy-associated virus(LAV), HTLV-III, or AIDS related virus (ARV), is a lentivirus from thefamily of retroviruses and is composed of RNA consisting of about 9,700base pairs, three gag proteins (having molecular weights of 55,000,24,000 and 17,000 daltons), a reverse transcriptase (molecular weightsof 66,000 and 51,000 daltons have been detected), three glycoproteins(two molecules having molecular weights of 120,000 and 41,000 daltons,and their precursor, a molecule with a molecular weight of 160,000daltons, hereinafter abbreviated as gp120, gp41 and gp160, respectively)which comprise the envelope, and other components. Exposed, envelopeproteins are particularly important for viral infection and thereforalso, the prevention thereof. As a result of proteolysis, gp1 60 iscleaved into gp120 and gp41. Gp4l is a transmembrane protein which isincorporated into the lipid bilayer of the viral envelope, while gp120is exposed on the outside of the envelope and some of it is releasedfrom the virus. Both gp4l and gp120 possess many sugar-binding sites,and about half of the gp120 molecule is comprised of sugars. The gp120molecule binds to the CD4 antigens on the surface of cells, inparticular helper T cells. Once HIV is bound to CD4 via gp 120, anotherenv gene product, gp41, mediates fusion between the membranes of thecell and the virus allowing the core of the virus to enter the cell.Gp120, which is expressed on the plasma membrane of infected cellsbefore virus is released, can bind to CD4 on another cell, initiating amembrane fusion event resulting in syncytia formation, and HIV genomescan be passed between the fused cells directly.

The env gene (gp120) is the primary determinant of cell tropism for bothHIV and Simian Immunodeficiency Virus (SIV). Variable region 3 (V3) ofgp120 is a key component within env that determines cell tropism. Theefficiency of replication and the ability to induce the syncytiaformation are also affected by changes in the V3 loop.

The first HIV virus isolated is referred to as HIV-1 and is generallydescribed in several articles, e.g., Barre-Sinoussi et al., Science220:868, 1983; Gallo et al., Science 224:500, 1984; Popovic et al.,Science 224:497, 1984; and Levy et al., Science 225:840, 1984, each ofwhich is hereby incorporated by reference. Various isolates of HIV-1have been obtained from North America, Western Europe and CentralAfrica. These isolates differ somewhat in their nucleotide sequence, butthe proteins they encode are generally antigenically cross-reactive.

A second virus related to HIV-1 has been isolated and termed HIV-2(Guyader et al., Nature 326:662, 1987; Brun-Vezinet et al., The Lancet1:128, 1987; and Clavel et al., Science 233:343, 1986). The geneticorganization of HIV-2 is similar to that of HIV-1. Of the two distinctsubtypes, HIV-1 is predominant and found throughout the world, whereasHIV-2 has been isolated primarily in West African countries such asGuinea Bissay, Ivory Coast, and Senegal, whith some cases alsoidentified in the Americas and western Europe. Epidemiological studiessuggest that the incubation period for HIV-2 for the development ofdisease is longer than for HIV-1.

HIV isolates from around the world were found to differ in nucleotidesequence. These sequences have been collected in a specialized database(Myers et al. (1994) Los Alamos National Laboratory, Los Alamos,N.Mex.). Two major groups of HIV has been identified. Viruses of group M(for "main") are responsible for the majority of infections worldwide;group O (for "outgroup") is a relatively rare group currently found inCameroon, Gabon, and France. Group M can be divided into at least eightdistinct subtypes or clades (A through H) (Myers, supra; Louwagie et al.(1995) J. Virol. 69:263). Isolates from HIV-1 from different clades maydiffer by 30-40% in the amino acid sequence of the gp120 SU protein;isolates within a lade vary from 5% to 20%. Clade B predominates inNorth America and Europe and lade E predominates in northern Thailand.Similarly, there are five HIV-2 sequence subtypes.

A group of viruses isolated from monkeys, termed simian immunodeficiencyvirus (SIV or STLV-III), is related to HIV-1 and HIV-2, particularly thelatter. See Daniel et al., Science 228:1201-1204 (1985); Kanki et al.,Science 230:951-954 (1985); Chakrabarti et al., Nature 328:543-547(1987); and Ohta et al., Int'l. J. Cancer 41:115-222 (1988). Members ofthis viral group exhibit minor variations in their genomic sequences,and have some differences in their restriction enzyme maps.

Although human CD4 is essential for HIV infection, it is not sufficient.Expression of human CD4 on rodent cells renders them capable of bindingvirus but still nonpermissive for fusion or infection (Maddon et al.(1986) Cell 47:333). The host component or coreceptors, sometimesreferred to as the "fusion receptors", were identified only recently.These coreceptors are receptors for chemokines (i.e. small proteinswhich serve as chemoattractants in inflammation) and they permit HIVinfection of virtually any mammalian or avian cell that expresses humanCD4 (Bates (1996) Cell 86:1-3). The most important coreceptors are CXCR4(also called "fusin" or "LESTR) (Endres et al. (1996) Cell 87:745; Fenget al. (1996) Science 272:872) and CCR5 (Akhatib et al. (1996) Science272:1955; Choe et al. (1996) Cell 85:1135; Deng et al. (1996) Nature381:661; Doranz et al. (1996) Cell 85:1149; and Dragic et al. (1996)Nature 381:667). CXCR4 is the receptor for the chemokine SDF-1, whereasCCR5 serves as a receptor for the chemokines MIP-1α and β and RANTES.These coreceptors play a crucial function for viral entry into cells,and they are also the principal determinants of tropism among CD4+cells.

Two distinct types of HIV-1 have been identified based on the cells inwhich they replicate in vitro. Viruses that replicate in T cell lines,but not macrophages or monocytes, are referred to as T tropic, whereasviruses with the complementary specificity are referred to as M tropic.The tropism is at least a function of the coreceptor: M tropic virusescan use only CCR5 for entry, and T tropic viruses use CXCR4. A few dualtropic isolates capable of using both are also known. T tropic virusesoften cause infected cells to fuse with uninfected cells if the latterexpress both human CD4 and CXCR4. Such viruses are referred to as'syncytium-inducing" (SI). All isolates can infect activated T cellsfreshly isolated from peripheral blood, which are present in PBMCcultures, since such cells express both CXCR4 and CCR5. Furthermore,cell tropisms are not fixed and can change when the virus is passaged incell culture (Metlzer et al. (1990) Immunology Today 11:217; Levy (1993)Microbiol. Rev. 57:183).

Two animal species (i.e., man and chimpanzee) are known to besusceptible to HIV infection, but only in man does the disease develop.HIV-1 transgenic mice carrying intact copies of the HIV-1 provirus havebeen obtained (Leonard et al. (1988) Science 242:1665). These micedevelop a spontaneous and fatal disease that mimics some of the featuresdescribed in human AIDS. Other HIV-1 transgenic mice carrying the HIV-1proviral DNA in which deletions have been introduced have also beenproduced (see, e.g., Dickie et al. (1991) Virology 185:109; Santoro etal. (1994) Virol. 201:147).

However, none of these transgenic mice closely model the development ofAIDS in humans. In particular, none of the HIV transgenic mice expressgp120 on the surface of their T cells. Thus, syncytium formation betweenHIV infected cells and CD4+ cells, e.g., T cells, which is reported tooccur in humans and which is in fact the mechanism by which HIV istransmitted from one cell to another without the production ofinfectious HIV particles, does not occur in HIV transgenic mice. Inaddition, since HIV transgenic mice do not express gp120 on the surfaceof infected cells and all of the neutralizing antibodies in humans havemapped to the envelope protein, gp160, or one of its component parts(gp120 or gp41), transgenic, HIV mice are not particularly useful fordeveloping human HIV vaccines.

Thus, there is a need for animal models of AIDS and other lentiviraldiseases, which more closely model infection and disease progression asit occurs in humans.

SUMMARY OF THE INVENTION

In one aspect, the present invention features non-human animal models oflentiviral (e.g., HIV) infection and development of disease (e.g. AIDS).Preferred non-human animals comprised of a lentiviral transgene arelarger than a mouse. Other preferred non-human, transgenic animals aresmaller than a monkey. A particularly preferred non-human, transgenicanimal is a transgenic rat.

In preferred embodiments, the transgene comprises essentially all of aviral genome, i.e., the transgene comprises at least about 70%, at leastabout 80%, at least about 90% or at least about 95% of a wild-type viralgenome. Also within the scope of the invention are transgenic animals inwhich the transgene comprises a smaller portion of the wildtype virus,(e.g. less than about 70% of the viral genome). For example, thetransgenic animal can comprise a transgene encoding a single protein.

In a preferred embodiment, the transgenic, non-human animal is comprisedof an HIV transgene. Exemplary HIV proteins for inclusion in thetransgene include an envelope protein (e.g., gp120 and gp40), a reversetranscriptase, a protease, an integrase, a ribonuclease, a nucleocapsidcore factor (gag), a transcriptional activator (e.g., tat and vpr) orproteins encoded by the genes vif, vpu, and nef

In another preferred embodiment, the transgenic, non-human animalexpresses the HIV proteins. In a particularly preferred embodiment, theanimal expresses the HIV protein gp120 on the surface of its peripheralblood mononuclear cells (PBMCs). In a further preferred embodiment, thetransgenic, non-human animal expresses at least one HIV coreceptor (e.g.CCR5 or CXCR4).

In another embodiment, in addition to the lentiviral transgene, thetransgenic, non-human animal is comprised of at least one additionaltransgene. In a preferred embodiment, the additional transgene is ahuman CD4 receptor gene. Expression by the animal of both HIV and humanCD4 allows the HIV particles produced to enter the CD4 expressing cells,thus resulting in HIV infection. In another preferred embodiment, theadditional transgene is an HIV coreceptor, such as CCR5 or CXCR4, whichfurther aids in HIV infection. In a further preferred embodiment, theadditional transgene is a gene involved in a disease or condition thatis associated with AIDS (e.g. hypertension, Kaposi's sarcoma, cachexia,etc.).

In another preferred embodiment, the non-human animal containing an HIVtransgene exhibits at least one symptom or phenotype characteristic ofhuman HIV infection and/or development of AIDS (e.g. development ofcataracts, cachexia or lesions (e.g. skin lesions, for example,resulting from psoratic dermatitis, hyperkerstotic lesions, kidneysclerotic lesions or inflammatory lesions of the central nervoussystem).

In another aspect, the invention features methods for producing thetransgenic animals described herein. A preferred method comprises thesteps of: (a) obtaining a non-human animal egg containing a lentivirustransgene; (b) implanting the egg of step (a) into a female non-humananimal; (c) selecting offspring containing the transgene to therebyobtain a founder animal; and (d) crossing the founder animal withanother animal preferably of the same species, but opposite sex, tothereby produce a transgenic animal comprising a lentivirus transgene.

In one embodiment, the lentiviral transgene, in step (a), is an HIVtransgene. In a preferred embodiment, the HIV transgene is infectious.For example, the transgene can be supplied by a wild-type HIV provirus,e.g, an HIV-1 or HIV-2 provirus or strain thereof Alternatively, thetransgene can be a modified form of a wild-type provirus, such as aprovirus having a deletion, substitution or addition of at least onenucleotide to the viral genome. For example, a provirus can be modifiedby replacing the transcriptional control element in an LTR of the HIVprovirus with another transcriptional control element, for example toalter the tropism of the virus. A provirus can also be modified bydeleting a portion of, or mutating, at least one HIV gene, to therebyinactivate at least one HIV protein, e.g, gag, pol, env, or tat. Inanother embodiment, the HIV transgene is non-infectious. For example, anHIV provirus can be rendered non-infectious by deleting a portion of gagand pol or by mutating at least one LTR of the provirus.

In a further aspect, the invention features non-human animal cellscontaining a lentivirus transgene, e.g., an HIV transgene. For example,the animal cell (e.g. somatic cell or germ cell (i.e. egg or sperm)) canbe obtained from a lentivirus transgenic animal. Transgenic somaticcells or cell lines can be used, for example, in drug screening assays.Transgenic germ cells, on the other hand, can be used in generatingtransgenic progeny, as described above.

In yet further aspects, the invention features methods for using thenon-human transgenic animals, cells and cell lines of the invention forinvestigating molecular and cellular mechanisms of lentiviral mediatedpathogenesis (e.g. the molecular and cellular mechanisms of the skinlesions, CNS disturbances, heart and kidney disease, which is associatedwith human HIV infection); as well as for identifying compounds andvaccines for treating and/or preventing lentivirus (e.g. HIV) infectionand disease (e.g. AIDS) development.

A preferred in vitro assay for identifying molecular antagonists which,for example, interfere with a lentivirus ligand-receptor interaction, aswell as molecular agonist which, for example, function by activating alentivirus protein (e.g. receptor) is comprised of the steps of: (a)incubating transgenic cells expressing a protein (e.g. receptor) knownto be involved in lentivirus infection with a test compound; and (b)detecting the interaction between the lentivirus protein and the testcompound, wherein the presence of an interaction indicates that the testcompound may be an inhibitor of lentivirus infection. In anotherembodiment, in step (a), the test compound is incubated with thetransgenic cell in the presence of a compound, which is a bindingpartner (e.g. a receptor ligand) to the expressed protein and theinteraction between the test compound and the lentivirus protein orbetween the lentivirus binding partner and the lentivirus protein isdetected.

In other embodiments, cell based assays can be used to identifycompounds which modulate expression of a lentivirus gene, modulatetranslation of a lentivirus mRNA, or which modulate the stability of alentivirus mRNA or protein. A preferred assay comprises the steps of:(a) incubating a transgenic cell, which expresses a particularlentivirus protein with a test compound; and (b) comparing the amount ofthe lentivirus protein produced to that produced by the same cell whichhas not been contacted with the test compound.

In a further embodiment, the effect of a test compound on transcriptionof a particular lentivirus gene can be determined by a transfectionassay, which uses a reporter gene operatively linked to at least aportion of the promoter of a lentivirus gene.

A preferred in vivo assay for identifying a compound which is useful fortreating or preventing a disease or condition associated with lentivirusinfection is comprised of the steps of. a) administering a test compoundto a lentivirus transgenic animal; and (b) observing at least onephenotype associated with infection by the lentivirus, wherein a changein phenotype indicates that the test compound is capable of treating orpreventing the disease or condition. In a preferred embodiment foridentifying an effective vaccine, the transgenic non-human animal ismade with an infectious lentivirus transgene, the compound is alentivirus antigen or combination of antigens and the phenotype is animmune response. In a particularly preferred embodiment for identifyingeffective HIV vaccines, the transgenic non-human animal is made with aninfectious HIV transgene, alone or in conjunction with a transgeneencoding a CD4 receptor (e.g. a human CD4 receptor) and/or an HIVco-receptor transgene (e.g. CCR or CXCR4).

In a further aspect, the invention features methods for treatingsubjects infected with a lentivirus or preventing infection by alentivirus, comprising administering to the subject an effective amountof a compound identified according to an assay of the invention.

Other aspects of the invention are described below or will be apparentto those skilled in the art in light of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram of the HIV-1 proviral DNA used to prepareHIV-1 transgenic rats as described in the Examples.

FIG. 2 shows the result of indirect immunofluorescence and flowcytometry analysis of peripheral blood mononuclear cells (PBMCs) ofHIV-transgenic rats, indicating that the PBMCs express gp120 on theirsurface (73.001: no staining; 73.002: Surface staining for env with F105human anti-env antibody plus anti-human-FITC secondary antibody; 73.004:Surface staining with anti-human-FITC secondary antibody alone).

DETAILED DESCRIPTION OF THE INVENTION

General

The invention is based at least in part on the generation of HIVtransgenic rats, which develop characteristic AIDS symptoms and thatexpress the HIV envelope protein, gp120.

Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples and appended claims are provided below.

"Antigen" refers to a protein, polypeptide, peptide or other molecule,which is capable of eliciting an immune response when administered to avertebrate.

"Animal line" refers to a group of animals that are direct descendantsof one founder animal and which bear one or more transgenes stablyintegrated into one or more loci in their germline.

A "DNA construct" refers to a DNA molecule comprising a transgene.

"Founder" generally refers to a first transgenic animal, which has beenobtained from any of a variety of methods, e.g., pronuclei injection.

"Genome" is intended to include the entire DNA complement of anorganism, including the nuclear DNA component, chromosomal orextrachromosomal DNA, as well as the cytoplasmic domain (e.g.,mitochondrial DNA). The genome of a eukaryotic organisms, in contrast tobacterial and viral organisms, is usually arranged into chromosomeswithin the cell nucleus.

"gp-120 is expressed on the surface of a cell" or "a cell expressinggp-120 on its surface" refers to a cell which contains at least onemolecule of gp-120 on its surface, preferably at least about 10, atleast about 100, at least about 1000, at least about 10,000 or at leastabout 100,000 gp-120 molecules on its surface. Preferred cellsexpressing gp-120 are those on which gp-120 can be detected by flowcytometry using an anti-gp120 antibody. Other preferred cells expressinggp-120 are cells which have a biological activity typical of cellshaving gp-120 on their surface, e.g., interaction with CD4 and/or whichare capable of syncytium formation.

"Heterologous DNA", which is used interchangeably with "exogenous DNA"refers to DNA that is not naturally present in the cell.

The term "HIV" is used interchangeably herein with the terms "LAV","LAV-2", "HTLV-III", and "ARV" to refer to human immunodeficiency virus(HIV). HIV includes both type 1 and type 2 human immunodeficiencyviruses and their strains, unless it is used within the context of aspecific embodiment related to type 1 or type 2 virus. The terms "HIV-1"and "HIV-2" are used to distinguish the type 1 virus and its strainsfrom the type 2 virus and its strains. The HIV-1 and HIV-2 genomes, andthe DNA sequences of HIV-1 and HIV-2, and respective strains are furtherdescribed herein, as well as in the publication "Human Retrovirus AndAIDS 1991", Eds. G. Myer et al., Theoretical Biology and Biophysics, LosAlamos National Laboratory, Los Almos, N. Mex., 87545, USA. Nucleotidesequences of HIV strains can be found in Genbank under the followingAccession Nos: 1) HIV-1: K03455, M19921, K02013, M38431, M38429, K02007and M17449; 2) HIV-2: M30502, J04542, M30895, J04498, M15390, M31113 andL07625 from J. M. Coffin, S. H. Hughes, and H. E. Varmus, "Retroviruses"Cold Spring Harbor Laboratory Press, 1997, p 804). In addition to HIVsequences available from Genbank as described above, HIV sequences canalso be found, e.g., in the HIV sequence database publically availableat hiv-web.lanl.gov. A map of the HIV-1 genome and transcripts can befound, e.g., in J. M. Coffin, S. H. Hughes, and H. E. Varmus,"Retroviruses" Cold Spring Harbor Laboratory Press, 1997, p803). Setforth in Table 1 is the name and nucleotide location of the major genesof HIV-1 (from Coffin et al., supra pp 802, 804):

                  TABLE 1                                                         ______________________________________                                        name nucleotides                                                                             comments                                                       ______________________________________                                        R     1-96     Repeat is a short sequence containing the                          transactivator response region (TAR, i.e., Tat                                Responsive)                                                                 U5  97-181                                                                    PBS 182-199                                                                   gag  336-1836 encodes Pr55 Gag                                                pro 1637-2099 encodes a Pr160 Gag-Pro-Pol precursor                           pol 2102-4640 pol gene products are synthesized as part of Pr160                            vif 4587-5163 encodes p23 Vif protein                           vpr 5105-5339 encodes p15 Vpr protein                                         tat 5377-5591 encodes p14 Tat protein; binds to the Tat                        7925-7968 region of R                                                        rev 5516-5591 encodes p19 Rev protein                                          7925-8197                                                                    vpu 5608-5854 encodes p16 Vpu protein                                         env 5771-8339 encodes the gPr160 Env precursor                                nef 8343-8710 encodes p27 Nef                                                 PPT 8615-8630 serves as principal primer for plus strand synthesis                          U3 8631-9085                                                    R 9086-9181                                                                 ______________________________________                                    

Amino acid sequences of HIV-1 proteins can also be found in Genbankunder the following Accession Nos.(from Coffin et al., supra p 804):gag-P04591; pol-P04585; env-p04578; vif-p03401; vpr-p05926; vpu-p05919;tat-p04608; rev-p04618; and nef-p0460 1. HIV-2 has basically the samemolecular organization as HIV-1. However, contrary to HIV-1, HIV-2contains a gene called vpx, which encodes a 14 kDa protein of unknownfunction. Another difference is that HIV-2 does not contain the vpugene, which is present in the HIV-1 genome. Another difference betweenHIV-1 and HIV-2 is the presence of a large insertion in the HIV-2 revgene. There are also significant differences between HIV-1 and HIV-2 revin addition to this insertion.

In addition, a human T cell line that produces HIV is available underATCC Designation No. CRL-8543. A vector containing the full length HIV-1genome is available under ATCC Designation No. 53069. DNA encodingspecific HIV genes is also available from the ATCC, e.g., a clone ofhuman TAR (HIV) RNA binding protein 1 is available under ATCCDesignation No. 107237 and a DNA encoding env-3 from HIV-1 is availableunder ATCC Designation No. 53072.

"Homology" or "identity" or "similarity" refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare identical at that position. A degree of homology or similarity oridentity between nucleic acid sequences is a function of the number ofidentical or matching nucleotides at positions shared by the nucleicacid sequences. Two DNA sequences are "substantially homologous" or"substantially similar" when at least about 75% (preferably at leastabout 80%, and most preferably at least about 90 or 95%) of thenucleotides match over the defined length of the DNA sequences.Sequences that are substantially homologous can be identified bycomparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra. A degree of identity of aminoacid sequences is a function of the number of identical amino acids atpositions shared by the amino acid sequences. A degree of homology orsimilarity of amino acid sequences is a function of the number of aminoacids, i.e. structurally related, at positions shared by the amino acidsequences. An "unrelated" or "non-homologous" sequence shares less than40% identity, though preferably less than 25% identity, with one of thesequences of the present invention. Two amino acid sequences are"substantially homologous" or "substantially similar" when greater than70% of the amino acids are identical, or functionally identical.Preferably, the similar or homologous sequences are identified byalignment using, for example, the GCG (Genetics Computer Group, ProgramManual for the GCG Package, Version 7, Madison, Wis.) pileup program.

An "infectious" virus or virus particle refers to a virus that iscapable of replicating and producing new viral particles when it infectsan appropriate cell.

An "inbred animal line" is intended to refer to animals which aregenetically identical at all endogenous loci.

The term "isolated" as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs, or RNAs,respectively, that are present in the natural source of themacromolecule. The term isolated as used herein also refers to a nucleicacid or peptide that is substantially free of cellular material, viralmaterial, or culture medium when produced by recombinant DNA techniques,or chemical precursors or other chemicals when chemically synthesized.Moreover, an "isolated nucleic acid" is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state. The term "isolated" is also used hereinto refer to polypeptides which are isolated from other cellular proteinsand is meant to encompass both purified and recombinant polypeptides.

"Lentiviruses" include primate lentiviruses, e.g., humanimmunodeficiency virus types 1 and 2 (HIV-1/HIV-2); simianimmunodeficiency virus (SIV) from Chimpanzee (SIVcpz), Sooty mangabey(SIVsmm), African Green Monkey (SIVagm), Syke's monkey (SIVsyk),Mandrill (SIVmnd) and Macaque (SIVmac). Lentiviruses also include felinelentiviruses, e.g., Feline immunodeficiency virus (FIV); Bovinelentiviruses, e.g., Bovine immunodeficiency virus (BIV); Ovinelentiviruses, e.g., Maedi/Visna virus (MVV) and Caprine arthritisencephalitis virus (CAEV); and Equine lentiviruses, e.g., Equineinfectious anemia virus (EIAV). All lentiviruses express at least twoadditional regulatory proteins (Tat, Rev) in addition to Gag, Pol, andEnv proteins. Primate lentiviruses produce other accessory proteinsincluding Nef, Vpr, Vpu, Vpx, and Vif. Generally, lentiviruses are thecausative agents of a variety of disease, including, in addition toimmunodeficiency, neurological degeneration, and arthritis. Nucleotidesequences of the various lentiviruses can be found in Genbank under thefollowing Accession Nos. (from J. M. Coffin, S. H. Hughes, and H. E.Varmus, "Retroviruses" Cold Spring Harbor Laboratory Press, 199,7 p804): 1) HIV-1: K03455, M19921, K02013, M3843 1, M38429, K02007 andM17449; 2) HIV-2: M30502, J04542, M30895, J04498, M15390, M31113 andL07625; 3) SIV:M29975, M30931, M58410, M66437, L06042, M33262, M19499,M32741, M31345 and L03295; 4) FIV: M25381, M36968 and Ul 1820; 5)BIV.M32690; 6)E1AV: M16575, M87581 and U01866; 6)Visna: M10608, M51543,L06906, M60609 and M60610; 7) CAEV: M33677; and 8) Ovine lentivirusM31646 and M34193. Lentiviral DNA can also be obtained from the AmericanType Culture Collection (ATCC). For example, feline immunodeficiencyvirus is available under ATCC Designation No. VR-2333 and VR-3112.Equine infectious anemia virus A is available under ATCC Designation No.VR-778. Caprine arthritis-encephalitis virus is available under ATCCDesignation No. VR-905. Visna virus is available under ATCC DesignationNo. VR-779.

"Nucleic acid" refers to polynucleotides such as deoxyribonucleic acid(DNA), and, where appropriate, ribonucleic acid (RNA). The term shouldalso be understood to include, as equivalents, analogs of either RNA orDNA made from nucleotide analogs, and, as applicable to the embodimentbeing described, single (sense or antisense) and double-strandedpolynucleotides.

"Non-infectious" virus or virus particle refers to a virus that isincapable of producing new viral particles even when it infects anappropriate cell. A non-infectious human immunodeficiency virustypically has a deletion in gag and/or pol and is thereby incapable ofreplicating and encapsidating the viral DNA.

"Phenotype" refers to an observable property of an organism (in contrastto the genotype, i.e. genetic composition of the organism).

The terms "protein", "polypeptide" and "peptide" are usedinterchangeably herein when referring to a gene product.

The term "proviral" DNA refers to a form of a virus that is integratedinto the genetic material of a host cell and by replicating with it canbe transmitted from one cell generation to the next.

"Small molecule" as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat inhibit viral gene expression, virus replication, and/or viralproduction.

DNA "regulatory elements" include transcriptional and translationalcontrol elements, such as promoters, enhancers, silencers, terminators,and the like, that provide for translation or expression of a nucleicacid. In eukaryotic cells, polyadenylation signals are controlsequences.

The phrase "therapeutically effective amount" as used herein refers toan amount sufficient to improve by at least about 15 percent, preferablyby at least about 50 percent, more preferably by at least about 90percent, and most preferably by about 100% (i.e., cure) a medicalcondition or symptoms thereof in a subject. Alternatively thetherapeutically effective amount can be an amount sufficient to reduceby at least about 15 percent, preferably by at least about 50 percent,more preferably by at least about 90 percent, and most preferably byabout 100% the viral load, expression of a gene, e.g, a viral gene, orviral replication.

A cell has been "transfected" by exogenous or heterologous DNA when suchDNA has been introduced into the cell. A cell has been "transformed" byexogenous or heterologous DNA when the transfected DNA effects aphenotypic change. Preferably, the transforming DNA should be integrated(covalently linked) into chromosomal DNA making up the genome of thecell.

The term "transgene" broadly refers to any nucleic acid that isintroduced into an animal's genome, including but not limited to genesor DNA having sequences which are perhaps not normally present in thegenome, genes which are present, but not normally transcribed andtranslated ("expressed") in a given genome, or any other gene or DNAwhich one desires to introduce into the genome. This may include geneswhich may normally be present in the nontransgenic genome but which onedesires to have altered in expression, or which one desires to introducein an altered or variant form. A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons, that may be necessary for optimal expression of a selectednucleic acid. A preferred transgene of the invention is a viraltransgene, e.g., a lentiviral transgene. A transgene can be as few as acouple of nucleotides long, but is preferably at least about 50, 100,150, 200, 250, 300, 350, 400, or 500 nucleotides long or even longer andcan be, e.g., an entire viral genome. A transgene can be coding ornon-coding sequences, or a combination thereof A transgene usuallycomprises a regulatory element that is capable of driving the expressionof one or more transgenes under appropriate conditions. A "lentiviraltransgene" refers to a nucleic acid comprising a nucleotide sequenceencoding at least one lentiviral protein or biologically active portionthereof. An "HIV transgene" refers to a nucleic acid comprising anucleotide sequence encoding at least one HIV protein or biologicallyactive portion thereof

A "transgenic animal" refers to any animal, preferably a non-humanmammal (e.g. mouse, rat, rabbit, squirrel, hamster, rabbits, guineapigs, pigs, micro-pigs, prairie, baboons, squirrel monkeys andchimpanzees, etc), bird or an amphibian, in which one or more cellscontain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell, directly or indirectly, byintroduction into a precursor of the cell, by way of deliberate geneticmanipulation, such as by microinjection or by infection with arecombinant virus. The term genetic manipulation does not includeclassical cross-breeding, or in vitro fertilization, but rather isdirected to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic animalsdescribed herein, the transgene causes cells to express a viral gene.However, transgenic animals in which the transgene is silent are alsocontemplated, as for example, the FLP or CRE recombinase dependentconstructs.

The term "treating" as used herein is intended to encompass curing aswell as ameliorating at least one symptom of the condition or disease.

A "vaccine" refers to a preparation containing at least one lentiviralantigen, which can be administered to a subject to produce orartificially increase immunity to a disease, which is caused by orcontributed to by a lentivirus. In addition to the at least one antigen,the vaccine can optionally comprise a pharmaceutically acceptablecarrier and/or an adjuvant.

The term "wild-type viral gene or genome" refers to a viral gene orgenome as it is found in nature, i.e., which has not been manipulated byman. Thus, there may exist several wild-type genomes for each type ofvirus.

Lentiviral Transgenes and Trans genic Animals Produced Therefrom

The invention provides for transgenic non-human animals comprising alentivirus transgene, (e.g., an HIV transgene). The lentiviral constructcan be an infectious virus, which is capable of replicating andproducing viral particles. An example of an infectious HIV-1 DNAincludes the DNA construct referred to as pNL4-3 (Adachi et al. (1986)J. Virol. 59:284 and Leonard et al. (1988) Science 242:1665; GenbankAccession No. M19921). Another infectious HIV-1 proviral construct ispNL4-32 (Strebel et al. (1987) J. Virol. 328:728 and Leonard et al.,supra). Transgenic non-human animals made with infectious HIVtransgenes, alone or in conjunction with a transgene encoding a CD4receptor (e.g. the human CD4 receptor) and/or an HIV co-receptortransgene (e.g. CCR5 or CXCR4) can produce infectious viral particles,which infect host cells, and therefore are particularly preferred fordeveloping effective HIV vaccines and therapeutics.

Non-human transgenic animals, which are noninfectious and thereforepotentially safer for use, can be generated using transgenes comprisedof non-infectious viral DNA, i.e., viral DNA which does not result inthe formation of viral particles upon infection of a host cell. Forexample, a non-infectious viral DNA can have a deletion or other type ofmutation in any coding region or regulatory region sufficient to impairviral nucleic acid replication, and/or assembly of virions. The deletioncan inhibit production of, or inactivate, one or more of the proteinsselected from the group consisting of a nucleocapsid-core factor (e.g.,gag), reverse transcriptase, protease, integrase, ribonuclease, andtranscriptional activator (e.g., tat). An example of an HIV-1 provirusthat is non-infectious is the pNL4-3 :dI443 vector, which is derivedfrom the infectious pNL4-3 vector by deletion of a 3.1 kb sequenceoverlapping gag and pol (sequences between the SphI and BalI sites atbases 1443-4556), but containing env and the other accessory genes tat,nef, vif, vpr, and vpu, together with the 5' and 3' long terminalrepeats (LTRs). As described in the following Examples, pNL4-3:d1443 hasbeen used to produce transgenic rats, which model human AIDS.

Other non-infectious HIV DNA can be obtained by deleting portions of oneor both LTRs. For example, the HIV DNA sequence can be prepared bydigesting a plasmid clone containing the DNA sequence of HIV-1 with arestriction enzyme that cleaves the HIV proviral DNA sequence at sitesproximal to its 5' and 3' ends, thereby removing essential controllingsequences, to yield a proviral DNA sequence truncated at both ends, sothat the eventual RNA expression from the cleaved fragment is renderednon-infectious, but still includes those elements required for theeventual production of at least some viral proteins. In other words, theHIV genome is modified to lack the sequences necessary for reversetranscription, integration and/or transcription. The extent to which the5' and 3' ends must be truncated to render the RNA non-infectious can bedetermined by standard methods (e.g. by transforming the fragment soobtained into a genomic equivalent of HIV-1 and testing the resultingvirus for cytopathic activity). As an example, the SacI restrictionenzyme can be used to cleave the pBH10 plasmid [B. H. Hahn et al.,Nature, 312, 166 (1984)] to yield an HIV-1 genome deleted of the 5' LTRand/or a portion of its 3' LTR. Non-infectious HIV proviral DNA deleted5' and/or 3' are further described in U.S. Pat. No. 5,574,206 byJolicoeur.

Another method for obtaining a non-infectious HIV proviral DNA sequenceinvolves truncating the HIV genomic DNA fragment from its 5' end to apoint on the untranslated 5' leader sequence located between about 50nucleotides downstream from the 5' LTR, but not including the nucleotidemarking the beginning of the splice donor sequence; and truncating thesame HIV DNA fragment from its 3' end sequence to a point locateddownstream of the nef gene, so that the complete encoding sequence ofthe nef gene is retained and sequences required for virus replication(i.e. the U5, R and part of the U3 sequences) are deleted.

Transgenic animals exhibiting tissue specific expression can begenerated, for example, by inserting a tissue specific regulatoryelement, such as an enhancer, into the viral transgene. For example, oneof the LTRs or a portion thereof can be replaced with another promoterand/or enhancer, e.g., a CMV or a Moloney murine leukemia virus (MLV)promoter and/or enhancer. For example, the proviral HIV DNA is pNL4-3,in which the two NF-κB binding motifs of the HIV core enhancer sequencesfrom the Moloney murine Leukemia Virus (Mo-MuLV) LTR by M13 mutagenesis(deleting nucleotides -129 to -74, with respect to the HIV mRNA cap siteand replacing them with nucleotides -365 to -40 from the Mo-MuLV). Thisconstruct is further described (as pHm4-3) in Dickie et al. (1996) AIDSRes. Human Retroviruses 12:177, which also describes transgenic micecontaining this construct.

An LTR of a proviral genome, e.g, HIV proviral genome, can also bereplaced with a mouse mammary tumor virus (MMTV) LTR, which is known tobe tissue specific toward various epithelial and hematopoietic tissues,some of which naturally support lentivirus (and especially HIV)replication. (for an example of such a construct, see, e.g., U.S. Pat.No. 5,574,206 issued Nov. 12, 1996 to Jolicoeur).

Alternatively, non-human transgenic animals that only express HIVtransgenes in the brain can be generated using brain specific promoters(e.g. myelin basic protein (MBP) promoter, the neurofilament protein(NF-L) promoter, the gonadotropin-releasing hormone promoter, thevasopressin promoter and the neuron-specific enolase promoter, see SoForss-Petter et al., Neuron, 5, 187, (1990). Such animals can provide auseful in vivo model to evaluate the ability of a potential anti-HIVdrug to cross the blood-brain barrier. Other target cells for whichspecific promoters can be used are, for example, macrophages, T cellsand B cells. Other tissue specific promoters are well-known in the art,see e.g. R.Jaenisch, Science, 240, 1468 (1988).

Non-human transgenic animals containing an inducible lentiviraltransgene (infectious or noninfectious) can be generated using inducibleregulatory elements (e.g. metallothionein promoter), which arewell-known in the art. Lentiviral gene expression can then be initiatedin these animals by administering to the animal a compound which inducesgene expression (e.g. heavy metals). Another preferred inducible systemcomprises a tetracycline-inducible transcriptional activator (U.S. Pat.No. 5,654,168 issued Aug. 5, 1997 to Bujard and Gossen and U.S. Pat. No.5,650,298 issued Jul. 22, 1997 to Bujard et al.).

Double, triple or multimeric transgenic animals, comprise at least oneother transgene in addition to the lentiviral transgene. In a preferredembodiment, the animal comprises an HIV transgene and a transgeneencoding human CD4 protein or a portion thereof sufficient for HIVinfection of target cells. The nucleotide sequence of cDNA encodinghuman CD4 can be found, e.g., in Maddon et al. (1985) Cell 42:93. Micecontaining a CD4 transgene are described, e.g., in Wang et al., (1994)Eur. J. Immunol., 24: 1553. A human CD4 transgenic rabbit has beendescribed, e.g, in PCT application No. PCT/FR93/00598 (WO 94/00568) byMehtali et al.

In another preferred embodiment, the animal comprises an HIV transgeneand a transgene, which expresses a coreceptor (e.g. CCR5 or CXCR4). In afurther preferred embodiment, the animal comprises an HIV transgene anda transgene (e.g. mutant gene), which is involved in a disease orcondition that is associated with AIDS (e.g. hypertension, Kaposi'ssarcoma, cachexia, etc.). For example, the transgenic animals of theinvention can be crossed with hypertensive rats of the transgenic ratstrain TGR(mREN2)27 harboring the murine Ren-2 gene or transgenic ratscomprising a transgene encoding human angiotensinogen and/or human renin(U.S. Pat. No. 5,731,489). These transgenic rats develop fulminanthypertension at an early age despite low levels of renin in plasma andkidney (described in, e.g., Lee et al. (1996) Am J Physiol 270: E919).

Where one or more genes encoding a protein are used as transgenes, itmay be desirable to operably link the gene to an appropriate regulatoryelement, which will allow expression of the transgene. Regulatoryelements, e.g., promoters, enhancers, (e.g. inducible or constitutive)or polyadenylation signals are well known in the art. Regulatorysequences can be endogenous regulatory sequences, i.e., regulatorysequences from the same animal species as that in which it is introducedas a transgene. The regulatory sequences can also be the naturalregulatory sequence of the gene that is used as a transgene.Accordingly, regulatory elements for a CD4 transgene can be the naturalCD4 regulatory elements and can include 5' flanking sequence of the CD4gene comprising promoter and enhancer sequences.

Alternatively, a transgene can be placed under the control of anexogenous regulatory element, i.e. a regulatory element, which is notthe normal regulatory element of the transgene. For example, a human CD4transgene can be placed under the control of a promoter that isfunctional in specific cell types, e.g., in T lymphocytes. An exemplarypromoter for use in transgenic rats is the rat CD2 promoter.Alternatively, the transcriptional regulatory element can be a viralpromoter, e.g, the MMTV LTR.

The transgene also preferably contains a polyadenylation signal (poly Aaddition sequence), which can comprise one or a tandem of two to four ofthe known poly A addition signal sequences, such as those derived fromthe SV40 genome, the casein 3' untranslated region or other 3'untranslated sequences known in the art. A convenient and readilyavailable source for the poly A addition signal is the commerciallyavailable pSV2neo vector from which the SV40 poly A addition signalsequence can be cleaved. The transgene can be prepared using techniquesknown in the art; for example see J. Sambrook et al., "MolecularCloning: A Laboratory Manual", 2nd ed, Vols 1 to 3, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA, 1989.

Constructs for use as transgenes can first be tested for expression incell lines. Where the transcriptional control elements in the constructare those from a virus, e.g., HIV, it may be desirable to use a testcell line of the same type as that which is naturally infected by thevirus. For example, when testing a construct derived from an HIVprovirus, it may be desirable to use a cell line in which HIV isexpressed and is preferably capable of replicating, e.g., T cell lines.Examples of cell lines in which HIV is known to replicate includeprimary human PBMC, isolated macrophages, isolated CD4+ T cells andcultured human cell lines, such as HeLa and H9. Expression of thetransgene and/or production of viral particles can be detected asfurther set forth herein.

Production of transgenic non-human animals

In general, transgenic animal lines can be obtained by generatingtransgenic animals having incorporated into their genome at least onetransgene, selecting at least one founder from these animals andbreeding the founder or founders to establish at least one line oftransgenic animals having the selected transgene incorporated into theirgenome.

Animals for obtaining eggs or other nucleated cells (e.g. embryonic stemcells) for generating transgenic animals can be obtained from standardcommercial sources such as Charles River Laboratories (Wilmington,Mass.), Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis,Ind.).

Eggs can be obtained from suitable animals, e.g., by flushing from theoviduct or using techniques described in U.S. Pat. No. 5,489,742 issuedFeb. 6, 1996 to Hammer and Taurog; U.S. Pat. No. 5,625,125 issued onApr. 29, 1997 to Bennett et al.; Gordon et al., 1980, Proc. Natl. Acad.Sci. USA 77:7380-7384; Gordon & Ruddle, 1981, Science 214: 1244-1246;U.S. Pat. No. 4,873,191 to T. E. Wagner and P. C. Hoppe; U.S. Pat. No.5,604,131; Armstrong, et al. (1988) J. of Reproduction, 39:511 or PCTapplication No. PCT/FR93/00598 (WO 94/00568) by Mehtali et al.Preferably, the female is subjected to hormonal conditions effective topromote superovulation prior to obtaining the eggs.

Many techniques can be used to introduce DNA into an egg or othernucleated cell, including in vitro fertilization using sperm as acarrier of exogenous DNA ("sperm-mediated gene transfer", e.g.,Lavitrano et al., 1989, Cell 57: 717-723), microinjection, genetargeting (Thompson et al., 1989, Cell 56: 313-321), electroporation(Lo, 1983, Mol. Cell. Biol. 3: 1803-1814), transfection, or retrovirusmediated gene transfer (Van der Putten et al., 1985, Proc. Natl. Acad.Sci. USA 82: 6148-6152). For a review of such techniques, see Gordon(1989), Transgenic Animals, Intl. Rev. Cytol. 115:171-229.

Except for sperm-mediated gene transfer, eggs should be fertilized inconjunction with (before, during or after) other transgene transfertechniques. A preferred method for fertilizing eggs is by breeding thefemale with a fertile male. However, eggs can also be fertilized by invitro fertilization techniques.

Fertilized, transgene containing eggs can than be transferred topseudopregnant animals, also termed "foster mother animals", usingsuitable techniques. Pseudopregnant animals can be obtained, forexample, by placing 40-80 day old female animals, which are more than 8weeks of age, in cages with infertile males, e.g., vasectomized males.The next morning females are checked for vaginal plugs. Females who havemated with vasectomized males are held aside until the time of transfer.

Recipient females can be synchronized, e.g. using GNRH agonist (GnRH-a):des-gly10, (D-Ala6)-LH-RH Ethylamide, SigmaChemical Co.,St. Louis, Mo.Alternatively, a unilateral pregnancy can be achieved by a briefsurgical procedure involving the "peeling" away of the bursa membrane onthe left uterine horn. Injected embryos can then be transferred to theleft uterine horn via the infundibulum. Potential transgenic founderscan typically be identified immediately at birth from the endogenouslitter mates. For generating transgenic animals from embryonic stemcells, see e.g. Teratocarcinomas and embryonic stem cells, a practicalapproach, ed. E. J. Robertson, (IRL Press 1987) or in Potter et al Proc.Natl. Acad. Sci. USA 81, 7161 (1984), the teachings of which areincorporated herein by reference.

Founders that express the gene can then bred to establish a transgenicline. Accordingly, founder animals can be bred, inbred, crossbred oroutbred to produce colonies of animals of the present invention. Animalscomprising multiple transgenes can be generated by crossing differentfounder animals (e.g. an HIV transgenic animal and a transgenic animal,which expresses human CD4), as well as by introducing multipletransgenes into an egg or embryonic cell as described above.Furthermore, embryos from A-transgenic animals can be stored as frozenembryos, which are thawed and implanted into pseudo-pregnant animalswhen needed (See e.g. Hirabayashi et al. (1997) Exp Anim 46: 111 andAnzai (1994) Jikken Dobutsu 43: 247).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals that carry thetransgene in some, but not all cells, i.e., mosaic animals. Thetransgene can be integrated as a single transgene or in tandem, e.g.,head to head tandems, or head to tail or tail to tail or as multiplecopies.

The successful expression of the transgene can be detected by any ofseveral means well known to those skilled in the art. Non-limitingexamples include Northern blot, in situ hybridization of mRNA analysis,Western blot analysis, immunohistochemistry, and FACS analysis ofprotein expression.

In particular, the expression of the gag protein (p55), the gag proteincleavage products p24 and p17, the envelope glycoprotein (gp160) and theenvelope protein cleavage product gp120 can be detected using specificprobes and/or antibodies. At least some of these antibodies arecommercially available. For example, monospecific anti-gp120 reagent canbe obtained, e.g., from Biochorm, Seromed Ref. D7324. Sheep anti-gp120serum of HIV-1 can also be obtained from the AIDS Research and ReferenceProgram (catalog no. 192), National Institutes of Health, Bethesda, Md.Human monoclonal anti-gp120 antibodies are also described in U.S. Pat.No. 5,695,927 issued Dec. 7, 1997 to Masuho et al.

Animal tissue can also be analyzed directly, e.g., by preparing tissuesections. In some embodiments, it is preferable to fix the tissue, e.g.,with paraformaldehyde or formalin. Tissue sections can be preparedfrozen, or can be paraffin embedded. Slides of animal tissue can be usedfor immunohistochemistry, in vitro hybridization or for regularhistology, e.g., hematoxylin and eosin staining.

Virus can be quantitated by reverse transcriptase (RT) activity, as iswell-known in the art. A change in viral load can also be determined byquantitative assays for plasma HIV RNA using quantitative RT-PCR asdescribed, e.g, in Van Gemen et al. (1994) J. Viro. Methods 49:157; Chenet al. (1992) AIDS 6:533. Alternatively, viral load can be determined byassays for viral production from isolated PBMCs. Viral production fromPBMCs is determined by cocultruring PBMCs from the subject with H9 cellsand subsequent measurement of HIV titers using an ELISA assay for p24antigen levels (Popovic et al. (1984) Science 204:497; PCT/US97/11202(W097/49373) by Gallo et al.). To identify lymphoid cell typesexpressing viral RNA, peritoneal inflammatory macrophages derived fromthe transgenic animals can be cultured ex vivo and examined by Northernblot analysis.

Disease Models and Drug Screening Assays

The invention further provides methods for identifying (screening) orfor determining the safety and/or efficacy of lentivirus therapeutics,i.e. compounds which are useful for treating and/or preventing thedevelopment of diseases or conditions, which are caused by, orcontributed to by lentiviral infection (e.g. AIDS). In addition theassays are useful for further improving known anti-viral compounds, e.g,by modifying their structure to increase their stability and/or activityand/or toxicity.

In vitro cellular assays

Cells from the transgenic animals of the invention can be established inculture and immortalized to establish cell lines. For example,immortalized cell lines can be established from the livers of transgenicrats, as described in Bulera et al. (1997) Hepatology 25: 1192. Celllines from other types of cells can be established according to methodsknown in the art."

In one cell-based assay, cells expressing a lentivirus protein (e.g.receptor) on the outer surface of its cellular membrane can be incubatedin the presence of a test compound alone or in the presence of a testcompound and a lentivirus protein binding partner (e.g. a receptorligand) and the interaction between the test compound and the lentivirusprotein or between the lentivirus binding partner (preferably tagged)and the lentivirus protein can be detected, e.g., using amicrophysiometer (McConnell et al. (1992) Science 257:1906). Aninteraction between the lentivirus protein and either the test compoundor the lentivirus protein binding partner can be detected, (e.g. with amicrophysiometer as a change in the acidification of the medium). Thisassay system thus provides a means of identifying molecular antagonistswhich, for example, function by interfering with a lentivirusligand-receptor interaction, as well as molecular agonist which, forexample, function by activating a lentivirus protein (e.g. receptor).

Cell based assays can also be used to identify compounds which modulateexpression of a lentivirus gene, modulate translation of a lentivirusmRNA, or which modulate the stability of a lentivirus mRNA or protein.Accordingly, a cell which is capable of expressing a particularlentivirus protein can be incubated with a test compound and the amountof the lentivirus protein produced in the cell medium can be measuredand compared to that produced from a cell which has not been contactedwith the test compound. The specificity of the compound for regulatingthe expression of the particular lentivirus gene can be confirmed byvarious control analyses, e.g., measuring the expression of one or morecontrol genes. This type of cellular assay can be particularly usefulfor determining the efficacy of antisense molecules or ribozymes.

In another embodiment, the effect of a test compound on transcription ofa particular lentivirus gene can be determined by transfectionexperiments using a reporter gene, which is operatively linked to atleast a portion of the promoter of a lentivirus gene. A promoter regionof a gene can be isolated, e.g., from a genomic library according tomethods known in the art. The reporter gene can be any gene encoding aprotein which is readily quantifiable, e.g, the luciferase or CAT gene.Such reporter gene are well known in the art.

In vivo assays in transgenic animals

In addition to providing cells for in vitro assays, the transgenicanimals themselves can be used in in vivo assays to identify lentiviraltherapeutics. For example, the animals can be used in assays to identifycompounds which reduce or inhibit any phase of the lentiviral lifecycle, e.g., expression of one or more viral genes, activity of one ormore viral proteins, glycosylation of one or more viral proteins,processing of one or more viral proteins, viral replication, assembly ofvirions, and/or budding of infectious virions.

In an exemplary embodiment, the assay comprises administering a testcompound to a transgenic animal of the invention and comparing aphenotypic change in the animal relative to a transgenic animal whichhas not received the test compound. For example, where the animal is anHIV transgenic animal, the phenotypic change can be the amelioration inan AIDS related complex (ARC), cataracts, inflammatory lesions in thecentral nervous system (CNV), a mild kidney sclerotic lesion, or a skinlesion, such as psoratic dermatitis, hyperkerstotic lesions, Kaposi'ssarcoma or cachexia. The effect of a compound on inhibition of Kaposi'ssarcoma can be determined, as described, e.g., in PCT/US97/11202(WO97/49373) by Gallo et al. These and other HIV related symptoms orphenotypes are further described in Leonard et al. (1988) Science242:1665.

In yet another embodiment, the phenotypic change is the number of CD4+ Tcells or the ratio of CD4+ T cells versus CD8+ T cells. In HIV infectedhumans as well as in HIV transgenic mice, analysis of lymph nodesindicate that the number of CD4+ T cells decreases and the number ofCD8+ T cells increases. Numbers of CD4+ and CD8+ T cells can bedetermined, for example, by indirect immunofluorescence and flowcytometry, as described, e.g., in Santoro et al., supra.

Alternatively, a phenotypic change, e.g. a change in the expressionlevel of an HIV gene can be monitored. The HIV RNA can be selected fromthe group consisting of gag mRNA, gag-pro-pol mRNA, vif mRNA, vpr mRNA,tat mRNA, rev mRNA, vpu/env mRNA, nef mRNA, and vpx mRNA. The HIVprotein can be selected from the group consisting of Pr55 Gag andfragments thereof (p17 MA, p24 CA, p7 NC, p1, p9, p6, and p2), Pr160Gag-Pro-Pol, and fragments thereof (p10 PR, p51 RT, p66 RT, p32 IN), p23Vif, p15 Vpr, p14 Tat, p19 Rev, p16 Vpu, gPr 160 Env or fragmentsthereof (gp120 SU and gp41TM), p27 Nef, and p14 Vpx. The level of any ofthese mRNAs or proteins can be determined in cells from a tissue sample,such as a skin biopsy, as described in, e.g., PCT/US97/11202(W097/49373) by Gallo et al. Quantitation of HIV mRNA and protein isfurther described elsewhere herein and also in, e.g., Dickie et al.(1996) AIDS Res. Human Retroviruses 12:1103. In a preferred embodiment,the level of gp120 on the surface of PBMC is determined. This can bedone, as described in the examples, e.g., by immunofluorescence on PBMCobtained from the animals.

For example, the proteins expressed in the cells of a transgenic animalof the invention may include processed gag proteins resulting from thecleavage of the HIV-1 encoded gag-pol gene, the cleavage being effectedby the HIV-1 encoded protease. Thus, in one embodiment, the inventionprovides a method for evaluating a test compound as a potential HIV-1protease inhibitor. In an exemplary embodiment, the method involves: (a)administering a test compound to the transgenic animal, and (b)examining the effect of the test compound on the expressed gag proteinsin the animal by monitoring the expression levels of the proteins orRNAs. The presence of the RNA transcript and the presence or decrease(or inhibition) of the processed proteins in the cells serves as a meansfor evaluating HIV protease inhibitors.

Likewise, since the presence of the gag and envelope proteins in thefluid and tissues of the transgenic animal denotes that the HIVregulatory protein, rev, is expressed, the present invention provides amethod for evaluating a test compound as a potential inhibitor of revfunction. In an exemplary embodiment, the method involves: (a)administering a test compound to the transgenic animal, and (b)examining the effect of the test compound on the expressed gag andenvelope proteins and the gag protein cleavage products in the animal bymonitoring the expression levels thereof

A further phenotypic change is the production level or rate of viralparticles in the serum and/or tissue of the animal. This can bedetermined, e.g., by determining reverse transcriptase (RT activity) orviral load as described elsewhere herein as well as in PCT/US97/11202(WO97/49373) by Gallo et al., such as by determining p24 antigen.

Yet another phenotypic change, which can indicate HIV infection or AIDSprogression is the production of inflammatory cytokines such as IL-6,IL-8 and TNF-α; thus, efficacy of a compound as an anti-HIV therapeuticcan be assessed by ELISA tests for the reduction of serum levels of anyor all of these cytokines.

A vaccine can be tested by administering a test antigen to a transgenicanimal of the invention. The animal can optionally be boosted with thesame or a different antigen. The production of viral particles orexpression of viral proteins is then measured at various times followingthe administration of the test vaccine. A decrease in the amount ofviral particles produced or viral expression will indicate that the testvaccine is efficient in reducing or inhibiting viral production and/orexpression. The amount of antibody produced by the animal in response tothe vaccine antigen can also be determined according to methods known inthe art and provides a relative indication of the immunogenicity of theparticular antigen.

Therapeutic and Prophylactic Compounds

Compounds identified above as being useful for preventing lentiviralinfection and/or treating a lentiviral disease, can be, e.g. a nucleicacid (e.g DNA, RNA or PNA), protein, peptide, peptidomimetic, smallmolecule, or derivative thereof Preferred compounds are capable ofbinding to, and inhibiting transcription, translation or processing of alentiviral RNA or protein. Examples include antisense, ribozyme ortriplex nucleic acids, small molecule ligands, antibody or antibody-likebinding fragments). Alternative compounds are competitive inhibitors ofa protein involved in lentiviral infection, such as a portion of humanCD4 sufficient to bind to gp120 and interfere with binding of gp120proteins on the surface of an infected cell or on the surface of a viralparticle to a human CD4 molecule on the surface of cells.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the Ld5O (The Dose Lethal To 50% Of ThePopulation) and the Ed₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀ /ED₅₀.Compounds which exhibit large therapeutic induces are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable delivery systems includemicrospheres which offer the possiblity of local noninvasive delivery ofdrugs over an extended period of time. This technology utilizesmicrospheres of precapillary size which can be injected via a coronarychatheter into any selected part of the e.g. heart or other organswithout causing inflammation or ischemia. The administered therapeuticis slowly released from these microspheres and taken up by surroundingtissue cells (e.g. endothelial cells).

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives, in addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In situations in which the therapeutic is a gene, a gene delivery systemcan be introduced into a patient by any of a number of methods, each ofwhich is familiar in the art. For instance, a pharmaceutical preparationof the gene delivery system can be introduced systemically, e.g., byintravenous injection, and specific transduction of the fir, protein inthe target cells occurs predominantly from specificity of transfectionprovided by the gene delivery vehicle, cell-type or tissue-typeexpression due to the transcriptional regulatory sequences controllingexpression of the receptor gene, or a combination thereof. In otherembodiments, initial delivery of the recombinant gene is more limitedwith introduction into the animal being quite localized. For example,the gene delivery vehicle can be introduced by catheter (see U.S. Pat.No. 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994)PNAS 91: 3054-3057). A therapeutic gene, such as a gene encoding anantisense RNA or a ribozyme can be delivered in a gene therapy constructby electroporation using techniques described, for example, by Dev etal. ((1994) Cancer Treat Rev 20:105-115).

A gene therapy preparation can consist essentially of a gene deliverysystem in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle or compound is imbedded.Alternatively, where the complete gene delivery system can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can comprise one or more cells which producethe gene delivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference. The practice of the presentinvention will employ, unless otherwise indicated, conventionaltechniques of cell biology, cell culture, molecular biology, transgenicbiology, microbiology, recombinant DNA, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature. See, for example, Molecular Cloning A Laboratory Manual,2^(nd) Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glovered., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis etal. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames &S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames &S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, AlanR. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986);B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., N.Y.); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1986).

EXAMPLES Example 1

Preparation of an HIV-1 transgenic rat

A transgenic rat containing a proviral HIV-1 DNA, i.e., plasmidpNL4-3:d1443, was prepared as follows.

The HIV-1 proviral DNA in the plasmid pNL4-3:d1443 (represented inFIG. 1) is an artificial recombination of two strains. The 5' half ofpNL4-3 is the viral isolate NY5 and the 3' half is the viral isolateLAV-1; the recombination splice occurred at the EcoRI site at nucleotide5743 base 1 being the most 5' nucleotide of the LTR). The DNA used forthe construction of the transgenic rat included a deletion of sequencebetween the BalI and SphI sites (nucleotides 1443-4551), therebydeleting the gag and pol genes. The construct included both the 5' and3' LTRs and open reading frames for the viral genes, env, tat, rev, nef,vif, vpr, and vpu. The upstream 5' splice acceptor site is left intact.Approximately 1 kb of human flanking sequence is present at both ends.Plasmid pNL4-3 has been described, e.g., in Leonard et al. (1988)Science 242:1665, illustrating the use of this plasmid for thepreparation of a transgenic mouse.

Specific pathogen-free inbred Fischer F-344/CrlBR (F344) rats, andoutbred Sprague-Dawley (SD) rats, were purchased from Charles RiverLaboratories, Boston, Mass. Animals were maintained in accordance withinstitutional guidelines.

Three week old Fisher 344 (F344) female rats (90-120 g) weresuperovulated according to Methods in Molecular Biology Vol. 18,Transgenesis Techniques, edited by David Murphy and David Carter, HumanaPress pp 253-256. Briefly, the female rats were injectedintraperitoneally with 0.2 IU/g body weight of pregnant mare serumgonadotropin (PMSG), followed 46-48 hours later with an intraperitonealinjection of 0.2 IU/g body weight of human chorionic gonadotropin (HCG).Each female was then placed in a cage with a stud male. On the morningof the next day, a check was made for copulatory plugs or vaginal smearswere performed to check for sperm in the lavage. At 12:00 that day,fertilized one-cell eggs were flushed from the oviducts of femalesexhibiting either a vaginal plug or sperm in vaginal lavage fluid. Themethod for collecting fertilized eggs was identical to that previouslydescribed for mice (Hogan et al. 1994 and Murphy et al. 1993). Two typesof culture media were used for in vitro manipulations of rat embryos.M16 was used for maintaining the eggs at 37° C. gassed with 5% CO₂. M2media was used for in vitro manipulations outside the CO₂ incubator forperiods less than 30 minutes. Eggs were collected from the ampulla in M2media containing 300 migcrograms/ml of hyaluronidase. Following removalof the cumulus cells, eggs were washed twice in fresh M2 and transferredto CO₂ equilibrated M16 and incubated at 37° C. until required formicroinjection.

Pseudopregnant rats were obtained as follows. Female SD rats, at least 8weeks of age, were maintained on a 12 hour day and 12 hour night cycleso that they ovulate and mate every 4 days. The stage of estrous wasdetermined prior to placing them with vasectomized rats. Sexually matureSD females were anasthetized with Methozyflurane, their vagina flushedand the contents dried and stained with a modified Wright's stain (e.g.,Dip-Quick). The vaginal contents were examined at 40× magnification andeach female was staged as to their position in the estrous cycle. SDfemales found to be proestrous were placed with vasectomized SD males,on day 0 by 18:00 hr to generate pseudopregnant recipients.

Vasectomized male rats were obtained by a surgical procedure (Hogan etal., supra). Male rates were anasthetized with 60 mg/kg Ketamine and 7.5mg/kg Xylazine. The abdomen was shaved and cleaned and the body wallincised and the left and right vas deferens were cauterized. The bodywall was then closed with wound clips and the rat caged individually ina warm place until recovered.

Microinjection of Fisher 344 X Sprague Dawley eggs and transfer to dayone pseudopregnant Sprague-Dawley females were carried out as follows.For injection, the eggs were transferred to M2 medium. Eggs weresequentially held in place by a blunt pipet (outside diameter about 100p) while the tip of the injector pipet was inserted through the zonapellucida and vitellus and into one of the pronuclei. The DNA solutionconsisting of plasmid pNL4-3: d1443 at a concentration of 2 ng/ml in theinjector pipet was slowly discharged by using a 100 μl Hamilton syringeconnected to a micrometer. The injector pipet was filled with siliconeoil except for the DNA solution. After injection, the eggs weretransferred to the oviducts of pseudopregnant Sprague-Dawley femalerats. The procedure was identical to that described for mice (E. Lacy etal., Manipulating the Mouse Embryo, Cold Spring Harbor Press, N.Y. 1994;and Methods in Molecular Biology Vol. 18, Transgenesis Techniques,edited by David Murphy and David Carter, Humana Press). Briefly, therecipient was anesthetized as previously described and the oviductsexteriorized by a surgical incision made at the level of the paralumbarfossa. Approximately 15-30 embryos were transferred per recipient. Thebody wall was then closed and the recipient was kept warm untilrecovery.

Potential founder transgenic rats were initially identified by PCRand/or by restriction enzyme digestion and Southern blot analysis. DNAfor PCR or Southern blot analysis was obtained from 2-3 weeks old rattail tips as per modification of the procedure of Hogan et al. (E. Lacyet al., Manipulating the Mouse Embryo, Cold Spring Harbor Press, N.Y.1994). Approximately, 1 cm long rat tail tips were excised with asterile scalpel following anesthesia with 0.02ml SQ of Lidocaine-HCL.Bleeding was controlled with silver nitrate. Following tail tipamputations, rats received Phenylbutazone 50 mg/kg, intraperitoneally asneeded for pain. A Quiagen kit was used to extract DNA from tail tips.

Identification and quantitation of transgenes was determined in thefounder animals and their progeny by Southern blot analysis of genomicDNA first amplified by PCR Two primers (SK68 (5' AGC AGC AGG AAG CAC TATGG; SEQ ID NO: 4) and SK69 (5° CCA GAC TGT GAG TTG CAA CAG; SEQ ID NO:5) were used to specifically amplify a 141 bp region from HIV-1 env byPCR-Southern blot hybridization with a ³² p labeled 1.2 kb HindIII NefcDNA fragment was used to confirm the identification of positiveanimals. Rats positive for the transgenic construct are referred to as"TgN(pNL43d14) FO1 MBC/IHV TG-1" rats. One female Sprague dawley xFisher 344/NHsd F1 rat was found to carry the HIV transgene. Thisfounder produced many hemizygous offspring and brother-sister matingsproduced further offspring.

Southern blot hybridization and PCR analysis indicates that copies ofthe proviral HIV genome are inserted in either 3-6 copies or 6-12 copiesin the genome. It is likely that the transgene is inserted on twodifferent chromosomes, resulting in transgenic animals having 3-6 copiesor 6-12 copies, depending on whether they have the two chromosomescarrying the transgene or only one. It is believed that the number ofcopies of the transgene correlates with the degree of certaincharacteristics of the phenotype of the animal, in particular with thedegree of cataracts (light verus heavy cataracts).

Example 2

Phenotype of the HIV transgenic rat

The female founder rat, TG-1, has cataracts in both eyes and a small redcircular lesion at the base of the tail. TG-1 was mated with a normalSprague Dawley male to produce the F₁ generation. The F₁ offsprings hadcataracts that varied from a high degree of opacity to a faint one. Thecataracts were supplied with a large number of blood vessels (highlyvascular). Transgenic animals had bilateral cataracts at birth. Twophenotypes could be distinguished: one phenotype which consisted ofheavy, dark cataracts, and the other consisted of light, mildercataracts.

In addition, at the time of weaning, most animals developed a focal skinlesion at the base of the tail. Of the 10 in the first litter three hadred lesions at the base of the tail, all were females. Subsequentoffsprings have also demonstrated that a few males have the red lesion.Mating of this phenotype produce offsprings with more severe skinlesions that cover the length of the tail and the offsprings weresmaller, especially the females. Early pregnancy in these animals causedthe lesions to disappear and return later in pregnancy and fullyreturned at partituation. The severe skin lesion phenotype were smaller,had a larger amount of proteins in urine, increased BUN and a higheralkaline urine. Skin lesions were that of psoratic dermatitis andhyperkerastotic lesions, mild kidney sclerotic lesions, and inflammatorylesions in the CNS. Thus, the HIV transgenic rat of the inventiondisplays many of the pathology seen in humans with HIV, includingretarded growth, CNS disturbances, mild to severe skin lesions, kidneyproblems and encephalitis.

Since the transgenic rat has a similar phenotype to that of a transgenicmouse containing the pNL4-3 proviral DNA, the phenotype of thetransgenic rat is most likely not due to an insertional inactivationevent.

Example 3

Expression pattern of HIV in HIV transgenic rats

Expression of HIV genes in the HIV transgenic rats obtained as describedin Example 1 was determined by RT-PCR as follows.

Rat tissues (i.e. eye, skin, muscle, brain, bone, heart, adrenal glands,kidney, large intestine, liver, lung, pancreas, small intestine, spleen,stomach, testicle, tongue, and thymus) were necropsied and snap-frozenin liquid nitrogen. The tissues were stored at -84° C. until processing.The tissues were homogenized in approximately 1 ml of Trizol (lifeTechnologies) using a PowerGen Homogenizer (Fisher Scientific). Afterhomogenation the samples were incubated at room temperature for 5minutes to permit the complete dissociation of nucleoprotein complexes.0.2 ml of chloroform was added to the samples before shaking for 2-3minutes by hand. The milky pink samples were then centrifuged at 12,000g for 15 minutes at 4° C. The mixture separated into a lower, red,phenol-chloroform phase, an interphase, and a colorless upper aqueousphase, which contains the RNA. The aqueous phase was transferred to afresh tube, and mixed with 0.5 ml isopropyl alcohol. The samples wereincubated for 10 minutes at room temperature then centrifuged at 12,000g for 10 minutes at 4° C. The RNA precipitate formed a pellet on theside and bottom of the tube. The supernate was removed from the pellet.The pellet was then washed once with about 1 ml of 75% ethanol. Thesample was then vortexed and centrifuged at 7,500 g for 5 minutes at 4°C. The supernate was once again removed, and the pellet was allowed toair-dry for up to 30 minutes. DEPC water was then added to redissolvethe pellet. To assist in dissolving the pellet, the samples wereincubated for 10 minutes (sometimes longer) at 60° C. The samples werethen stored at -80° C.

cDNA was prepared from the RNA as follows. 20 μl of the RNA sample wasincubated with 20 U of DNAse I (10 Units/μl) for 1 hour at 37° C. Thesample was then phenol extracted (Trizol method above) to remove the DNAand DNAse protein from the RNA. The RNA was precipitated with ethanoland sodium acetate overnight. The pellet was washed, and dissolved asdescribed above. 2 μl of the sample was diluted with 200 μl of deionizedwater. The sample was then quantitated to determine the amount of RNApresent. 17 μl was made to contain 2 μg, of Dnased RNA. On ice, acocktail of 1 l Random Heximers 100 μM, 6 μl 5× Reverse TranscriptaseBuffer, 3 μl DTT 0.1 M, 1.5 μl DNTP 10 mM, 0.5 μl RNAse inhibitor, and 1μl Reverse Transcriptase (200 u) Moloney Murine Leukemia Virus ReverseTranscriptase was added to the Dnased RNA. For samples reversedtranscribed with the Art7 primer, 1.6 μl Art7 and 5.4 μl 5× RT bufferwas used. The mixture was incubated for 1 hour at 37° C. The sampleswere then heated at 95° C. for 5 minutes to kill the enzyme. The sampleswere immediately put on ice.

PCR was performed as follows. 5 μof cDNA was added to 40 μl of PCRSuperMix (Gibco). The PCR SuperMix contains 22 mM Tris-HCl (pH 8.4), 55mM KCl, 1.65 mM MgCl₂, 220 μM dGTP, 220 μM dATP, 220 μM dTTP, 220 μMdCTP, 22 U recombinant Taq DNA Polymerase/ml, stabilizers. 5 μl of 5',3' primers (20 μM) were added to the reaction mixture. The samples werefirst denatured for 3 minutes at 95° C. The parameters for PCRamplification were as follows: 35 cycles, each with denaturation at 95°C. for 1 minute, annealing at 60° C. for 2 minutes, and extension at 72°C. for 2 minutes. The final cycle was followed by a 5 minute extensionat 72° C. The samples were then held a 4° C.

The sequences of the primers and probes used for cDNA synthesis anddetection as well as the expected products are as described in Bruggemanet al. (1994) Virology 202:940. Since the mRNA encoding all HIV-1proteins are processed from the same precursor RNA with alternativesplicing and all HIV-1 mRNAs have identical 5' exons, the 5' senseprimer used for all cDNA synthesis was US (TAG TAG CAT GCT CTC TCG ACGCAG GAC TCG GCT TGC; SEQ ID NO: 1). The primer pair US and ART7 (ATG ATCTGC AGT TCT ATT CCT TCG GGC CTG TCG; SEQ ID NO: 3) was used to amplifythe tat, rev, and nef genes. Probing of the amplified products with S1identifies a 402 bp tat fragment, while probing with S2 identifies 402bp-tat and 219/225 bp-rev fragments and probing with S3 identifies 402bp-tat, 219/225 bp-rev and 203 bp-nef fragments. The primer ART5 isdownstream from the slice acceptor for vif and the primer ART2 isdownstream from the initiation codon of Env. The primer pair ART5/USamplifies vif mRNA and generates a 338 bp fragment when probed with S4.Primers ART2 (ACC TCC TGC AGC ACA GGT ACC CCC ATA ATA GAC TGT G; SEQ IDNO: 2) and US was used to amplify env mRNA and generated a 446 and 665bp product when probed with the S3 probe. The 5' and 3' primers forG3PDH and SK68 (5' AGC AGC AGG AAG CAC TAT GG; SEQ ID NO: 4) and SK69(5' CCA GAC TGT GAG TTG CAA CAG; SEQ ID NO: 5) for Env were used toamplify regions of cDNA generated from random hexamers.

Following amplification, the reaction mixtures were subjected toelectrophoresis, the nucleic acids were transferred onto a blot and theblot was hybridized with the following probes:

S1 GAG CCA GTA GAT CCT AGA CTA GAG C (SEQ ID NO: 6);

S2 CTT AGG CAT CTC CTA TGG CAG GAA (SEQ ID NO: 7);

S3 ACC TCG CAT GCG AAG AAG CGG AGA CAG CGA CGA AG (SEQ ID NO: 8); and

ENV TGA CGC TGA CGG TAC AGG CC (SEQ ID NO: 9).

The results of tissue expression analysis indicate the presence oftranscripts of about 7 kb (full length gag-pol mRNA), 4 kb (singlyspliced env. mRNA) and 2 kb (multiply spliced tat, rev, and nef mRNA) innumerous tissues including the eyes, skin, and muscle and moderateexpression in the brain and heart, and light expression in bone andbladder. No detectable levels of Env mRNA were found in the adrenalglands, kidney, large intestine, liver, lung, pancreas, small intestine,spleen, stomach, testicle, tongue, and thymus.

Example 4

gp120 is present in the serum and on the surface of PBMCs in HIVtransgenic rats

Expression of the envelope protein in serum and PBMC was assayed byELISA capture and flow cytometry, respectively. The ELISA assayindicated that two of the hemizygous transgenic animals containing gp120in their sera at levels of approximately 145 pg/ml.

Multicolor flow cytometry of PBMCs was performed on a FACSCalibur(Becton-Dickinson Mountain View, Calif) as previously described (Taurogand El-Zaatari (1988) J Clin Invest 82, 987-92; Taurog et al. (1988) JClin Invest 82, 987-92). Briefly, Ficoll-Hypaque purified peripheralblood mononuclear cells were incubated with saturating concentrations ofF105 human anti-env antibody (available from the AIDS Repository),washed, then incubated with anti-human-FITC secondary antibody(available from Pharmigen Commercial). The staining was done in thepresence of 1% human AB serum (available from Sigma Commercial). Afterwashing, the cells were fixed in 1% paraformaldehyde before analysis ona FACScan flow cytometer (Becton Dickinson, Mountain View, Calif).Viable lymphocytes were selected for analysis by gating of forward and90° light scatter.

The FACS analysis, which is shown in FIG. 2, indicates that gp120 wasreadily detectable on the surface of the entire PBMC population. Thus,the data indicate that, contrary to mice transgenic for pNL4-3:d1443,the gp120 env protein is expressed on the surface of PBMCs of IRVtransgenic rats and shed into their serum.

Example 5

Production of an HIV/human CD4 double transgenic rat

A rat transgenic for HIV, e.g, HIV-1 and human CD4 can be prepared bycrossing an HIV-1 transgenic rat described in the previous examples witha rat containing a human CD4 transgene and selecting for offspringcarrying both transgenes.

A human CD4 transgenic rat can be prepared as follows. A constructcontaining human CD4 transgene, such as pES/CD4 (Louis Flamand; hCD4 isalso described in Gillespie et al. (1993) Mol. Cell. Biol. 13:2952),containing a 6 kb Mlu 1-Sal 1 fragment (ES/CD4) with the CD4 structuralgene and its regulatory sequence can be linearized and used to preparetransgenic rats using the technique of pronuclei injection describedabove for the preparation of the HIV transgenic rat.

The offspring is tested for the presence of the HIV transgene asdescribed above and for the presence of the human CD4 transgene usingthe PCR primers that are specific for the human CD4 gene and which donot interact with the rat CD4 gene or by Southern blot hybridizationusing a probe hybridizing specifically to human and not to rat CD4.Expression of the transgene can be determined by Northern blot and/orflow cytometry or FACS analysis, as described above. For example, todetect hCD4 expression, PBMC can be resuspended at a concentration of10⁷ cells per ml in cold PBS with 2% serum on ice. A total of 10⁶ cellscan then be reacted with anti-human and anti-rat CD4 antibody conjugatedwith APC fluorochrome and subjected to analysis by flow cytometry. Ithas been observed previously that human and rat CD4 antibodies do notcross react.

Infection of hCD4 transgenic rats can be performed as follows. Mature (6to 8 weeks old transgenic rats can be inoculated either intravenously(IV) or intraperitoneally (IP) with various concentrations of HIV (IIIB)(0.1-20 TCID₅₀) or with 10⁵ HIV-1 (IIIB)-infected CEM cells. Controlanimals can be non transgenic rats injected with non-infectious virusand hCD4 transgenic rats infected with the NSI HIV-1 (BA-L) or withdiluted pellets from non-infected CEM cells. The presence of HIV-1antibodies and viral antigen (p24) in the sera can then be analyzedevery 2 weeks for the first two months and at 4 months post inoculationusing a commercially available ELISA test. Rat PBMCs can be isolated onFicoll-hypaque and 1.0×10⁶ cells can be cultured with 0.3×10⁶ CEM cells.Simultaneously, 10⁶ PBMC can be treated with 3 μg/ml of PHA overnightand then cultured with CEM. Cultures can be examined for CPE for 1 monthand supernatant can be checked for antigen production by ELISA weekly.

Infection by HIV can also be tested in rats which are also trangenic fora construct containing the HIV-1 LTR upstream of DNA encoding the greenfluorescent protein (GFP), for example. Infection of a cell containingthis construct will result in stimulation of the LTR and therefore theGFP.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - TAGTAGCATG CTCTCTCGAC GCAGGACTCG GCTTGC      - #                       - #       36                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ACCTCCTGCA GCACAGGTAC CCCCATAATA GACTGTG      - #                      - #      37                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - ATGATCTGCA GTTCTATTCC TTCGGGCCTG TCG       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AGCAGCAGGA AGCACTATGG            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - CCAGACTGTG AGTTGCAACA G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                - - GAGCCAGTAG ATCCTAGACT AGAGC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - CTTAGGCATC TCCTATGGCA GGAA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - ACCTCGCATG CGAAGAAGCG GAGACAGCGA CGAAG       - #                  -     #       35                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: other nucleic acid                                         (A) DESCRIPTION: /desc - #= "primer"                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - TGACGCTGAC GGTACAGGCC            - #                  - #                      - # 20                                                                 __________________________________________________________________________

What is claimed is:
 1. A transgenic rat, whose genome contains at leastone copy of a human immunodeficiency virus type 1 (HIV-1) proviral DNAwherein the gag and pol genes are mutated to render the proviral DNAnoninfectious, and wherein said rat develops at least one symptom ofAcquired Immune Deficiency Syndrome (AIDS).
 2. The transgenic rat ofclaim 1, wherein the HIV-1 proviral DNA has been introduced into saidrat's genome via the vector pNL4-3:D1443.
 3. The transgenic rat of claim1, wherein HIV-1 gp120 protein is present on the surface of said rat'speripheral blood mononuclear cells (PBMCs).
 4. The transgenic rat ofclaim 3, wherein the HIV-1 gp120 protein is also present in the rat'sblood.
 5. An isolated cell of the transgenic rat of claim
 1. 6. The cellof claim 5 wherein the cell is a germ cell.
 7. The cell of claim 5,wherein the cell is a somatic cell.
 8. An isolated egg of the transgenicrat of claim
 1. 9. An assay for indentifying a compound which reduces atleast one symptom of AIDS in a subject, comprising the steps of:(a)administering a test compound to an HIV-1 transgenic rat of claim 1; and(b) determining the severity of at least one symptom of AIDS in thetransgenic rat of step (a), such that a reduction in the severity ofsaid at least one symptom of AIDS in the transgenic rat to which thecompound was administered relative to the severity of the said at leastone symptom of AIDS in said transgenic rat before the compound wasadministered indicates that the compound reduces said at least onesymptom of AIDS.
 10. The assay of claim 9, wherein said at least onesymptom of AIDS is selected from the group consisting of skin lesions,kidney lesions, cataract, and central nervous system (CNS) disturbances.11. The assay of claim 9, wherein said at least one symptom of AIDS isthe presence of HIV-1 gp120 on the PBMCs of the transgenic rat.
 12. Theassay of claim 9, wherein said at least one symptom of AIDS is thepresence of HIV-1 gp120 in the blood of the transgenic rat.
 13. A methodof generating the transgenic rat of claim 1, comprising introducing intoan embryo of a rat a HIV-1 proviral DNA (HIV-1) wherein the gag and polgenes are mutated to render the proviral DNA non-infectious, andpermitting the embryo to develop into a rat containing said HIV-1proviral DNA in its genome.